Genome Sequences of 18 Salmonella enterica Serotype Hadar Strains Collected from Patients in the United States

ABSTRACT Despite being linked to a number of recent poultry-associated outbreaks in the United States, few reference genomes are available for Salmonella enterica serotype Hadar. Here, we address this need by reporting 18 Salmonella Hadar genomes from samples collected from patients in the United States between 2014 and 2020.

S almonella enterica serotype Hadar infections in humans in the United States increased in 2020 and 2021, compared with previous years, despite an overall decline in reported salmonellosis cases (1). Many infections occurred as part of recent outbreaks linked to either backyard poultry flocks (e.g., chickens and ducks) or consumption of ground turkey, but isolates linked to these different sources demonstrated a high degree of core genome relatedness (1,2). Exploring the accessory genome may improve strain differentiation, as well as our understanding of the recent increase and evolution of this serotype. Here, we generated assemblies for 18 S. Hadar isolates collected from U.S. patients to serve as references for future investigations.
Isolates were selected for long-read sequencing based on diverse accessory genome content. Genomic DNA was extracted (Wizard genomic DNA purification kit [Promega, Madison, WI, USA], with a modification of the manufacturer's protocol) from cultures that had been incubated on tryptic soy agar-sheep blood overnight at 37°C. Libraries were prepared using the rapid barcoding kit (SQK-RBK004; Oxford Nanopore Technologies [ONT], Oxford, UK) according to the manufacturer's protocol and sequenced for 72 h on a GridION sequencing platform (R9.4.1 flow cells; ONT). Reads were base called using Guppy v4.2.2 and filtered for quality using MinKNOW (ONT). Hybrid assemblies were generated, polished, circularized, and rotated using Unicycler v0.4.8 (conservative option) (8); corresponding Illumina short reads that had been previously generated at the PHL (BioNumerics v7.6 [Applied Maths NV,  Plasmid assigned using updated version of COPLA.
c Plasmid replicon missing from long-read assembly but present in short reads.

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Sint-Martens-Latem, Belgium] quality control metrics: quality score, $30; coverage, $30Â) were accessed through NCBI (Table 1). Assemblies were quality controlled using QUAST v5.0.2 (9) and BLASTn v2.9.0 (10) and were annotated using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) v6.1 (11). Default parameters were used for all software unless otherwise specified. All 18 S. Hadar strains were found to be ST33. Resistance determinants and plasmid types are summarized in Table 1. The most common resistance genes were aph(30)-Ib, aph(6)-Id, and tet(A), which were always located on the chromosome (n = 13). When present, other resistance genes were associated with IncI1-I g or Col(pHAD28) plasmids. High levels of small plasmids with no known resistance genes were observed, some of which had not been previously characterized, as indicated by small, circular genetic elements not containing a known plasmid replicon. More generally, the hybrid assembly method employed here recovered small plasmids at a higher rate than did long-read-only assembly methods (data not shown). For two genomes, however, some small plasmids were not recovered despite the use of a hybrid assembly method (Table 1), a known artifact of the long-read sequencing process (12).
Data availability. The sequences discussed here have been deposited in GenBank and the SRA under the accession numbers listed in Table 1.